Jamel Jebali

Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast

Abstract In mussel Mytilus galloprovincialis tissues, metallothionein belongs to two different gene classes, mt10 and mt20, showing differential expression at both basal conditions and under heavy metal challenge. In this study, a new more highly sensitive technique, expression analysis of mt10 and mt20 mRNA levels by quantitative reverse transcription polymerase chain reaction, was used to assess the effects of heavy metal contamination in the digestive glands of mussels caged along the Tunisian coast. To validate the new assay, total metallothionein protein, amount of heavy metals (zinc, copper, cadmium), and a biomarker of oxidative stress such as malondialdehyde content, were assessed in the same tissues. At the investigated sites, the molecular assay showed variations of mt20 relative gene expression levels within one or two orders of magnitude, with maximum values at two sites severely polluted with cadmium, Mahres (100-fold) and Menzel Jemile (165-fold). Changes in mt10 expression were recorded at all sites where copper had significantly accumulated, although fold induction levels were less pronounced than those of mt20. In this paper, gene expression data are discussed in relation to the studied biomarkers, demonstrating that the molecular technique based on the differential expression of mt10 and mt20 genes represents (i) a useful and robust tool for studying and monitoring heavy metal pollution under field conditions, and (ii) an improvement in the application of metallothionein as a biomarker of response to exposure to heavy metals in marine mussels.

Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers

Abstract Over the past decade, molecular, biochemical and cellular markers have been extensively used in pollution monitoring of aquatic environments. Biochemical markers have been selected among early molecular events occurring in the toxicological mechanisms of main contaminants. This paper assesses the marine environment quality along the Tunisian coasts using a statistical approach. Clams (Ruditapes decussatus) were collected during the four seasons of 2003 on seven different sites from the Tunisian coasts. Oxidative stress was evaluated in gills using catalase activity (Cat), neutral lipids and malonedialdehyde accumulation. Glutathione S-transferase activity is related to the conjugation of organic compounds and was evaluated in both, gills and digestive glands. Acetylcholinesterase activity was evaluated as the biomarker of exposure to organophosphorous, carbamate pesticides and heavy metals. For each biomarker, a discriminatory factor was calculated and a response index allocated. For each site, a global response index was calculated as the sum of the response index of each biomarker. Discriminant analysis shows significant differences between sites and seasons compared with control sample. Faroua (site 1) and Menzel Jemile (site 2) seem to be the less polluted with respect to the other sites for all seasons. Gargour (site 6) shows the highest Multimarker Pollution Index during the four seasons, indicating higher contamination level.

Acute effects of benzo[a]pyrene on digestive gland enzymatic biomarkers and DNA damage on mussel Mytilus galloprovincialis

In the present study, mussel (Mytilus galloprovincialis) digestive gland biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) were investigated. Mussels were exposed to a sublethal dose of B[a]P (75 nM; 19 microg/l per animal) for 24, 48 and 72h. The following biological responses were measured in the digestive gland tissues: (1) B[a]P hydroxylase (BPH) activity, as phase I biotransformation parameter; (2) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (3) catalase (CAT) activity as potential biomarker of oxidative stress, (4) acetylcholinesterase (AChE) activity as an indication of possible neurotoxicity response. DNA damage was assessed over time using the single cell gel electrophoresis comet assay and the micronuclei test. BPH and GST activities showed an increasing trend over exposure period. CAT activity showed a symmetrical bell shape response with a maximum at 48h. AChE activity was significantly depressed after 48 and 72h exposure to B[a]P. Comet assay and micronuclei test in digestive gland cells suggest that B[a]P exposure induced significant DNA damage with a maximum response after 72h exposure.

Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations

In the present study, mussel (Mytilus galloprovincialis) digestive gland oxidative stress biomarkers and detoxification responses to acute exposure to nickel (Ni) were investigated. Mussels were exposed to two sublethal concentrations of Ni (135 μg/L per animal (2.5 μM) and 770 μg/L per animal (13 μM)) for 24, 48, 72, 96 h and 8 days. Following biological responses were measured: (1) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (2) catalase activity as antioxidant response, (3) malondialdehyde accumulation (MDA) as lipid peroxydation marker and metallothionein as specific response to metals exposure. The cholinergic system was evaluated using the acetylcholinesterase activity (AChE). Moreover, Ni uptakes during the exposure periods were assessed and the uptake rate constant determined. A correlation matrix (CM) between the investigated biomarkers and a principal component analysis (PCA) were achieved for the two tested concentrations. The Ni-uptake constant was higher in animals exposed to the lowest concentration. The CM and the PCA showed a time-dependent effect of the Ni exposure on the investigated biomarkers being more pronounced in animals exposed to the highest Ni concentration. While AChE showed a significant increase after 48 h and a further return to control values in the lowest concentration, it was drastically maintained inhibited in the highest concentration. Our data provided clues about the occurrence of different toxicokinetics and toxicodynamics of two Ni sublethal concentrations in an ecologically relevant organism.

Multimarker approach analysis in common carp Cyprinus carpio sampled from three freshwater sites.

The aim of this study is to assess the response of a multimarker approach in common carp Cyprinus carpio sampled from three Tunisian dam lakes selected according to different environmental and ecological characteristics. Glutathione-S-transferase (GST) activity was analyzed in carp liver and used as a phase II detoxification enzyme, hepatic metallothionein content (MTs) was used as a metallic stress indicator, and cholinesterase activities were analyzed in muscle and brain and used as neurotoxicity biomarker. Micronucleus frequency test (MN) as a genotoxicity marker. GST and MT levels showed an increase in fish from the Bir Mcherga site and a decrease in Sidi Saâd site with respect to fish from Nebhana site. Results showed a strong inhibition of cholinesterase activities in fish from Bir Mcherga and Sidi Saâd sites compared to Nebhana site. Relatively high level of MN is reported specially in fish blood from the Bir Mcherga site.

Metallothionein induction by Cu, Cd and Hg in Dicentrarchus labrax liver : Assessment by RP-HPLC with fluorescence detection and spectrophotometry

Metallothionein was quantified in sea bass Dicentrarchus labrax intraperitoneally (i.p.) injected with different Cu, Cd and Hg doses (50-250 microg kg(-1) wet wt) after 48 h exposure. A distinct peak with 16.8 min retention time was obtained by reversed-phase high performance liquid chromatography coupled to fluorescence detection (RP-HPLC-FD) with the three metals. Total metallothionein levels assayed in unheated liver extracts by RP-HPLC-FD were significantly higher (1.3-1.95-fold) than those obtained by the well-established spectrophotometric method. In the RP-HPLC-FD method, metallothionein increased linearly with Cu and Hg doses, being saturated beyond 100 mug kg(-1) Cd. Maximum induction was obtained at 100 microg kg(-1) Cd (5.3-fold), and 250 microg kg(-1) Cu or Hg (8- and 5.1-fold, respectively). At low doses no metallothionein induction was shown by the less sensitive spectrophotometric assay.

Biochemical effects in crabs ( Carcinus maenas ) and contamination levels in the Bizerta Lagoon: an integrated approach in biomonitoring of marine complex pollution

The biochemical effects in Carcinus maenas and contamination levels in seawater and sediments of Bizerta Lagoon (northeast of Tunisia) were investigated. The levels of metals and hydrocarbons were higher in seawater and sediments in Menzel Bourguiba and Cimentery in February and July than in the other sampling sites. Differences among sites for glutathione S-transferase, catalase, acetylcholinesterase activities, and the content of lipid peroxidation and metallothioneins in two important organs which accumulated contaminants (the gills and the digestive gland) of the C. maenas were found and possibly related to differences in metal and hydrocarbon levels. The seasonal variation of biomarkers was possibly associated with chemical contamination and also with the high fluctuation of physico-chemical characteristics of the sites. The integrated biomarker response values found in the five sites is in good agreement with hydrocarbon and trace metal concentrations detected in the water and sediments of the stressful places where crabs are living.

Seasonal variation of oxidative stress biomarkers in clams Ruditapes decussatus sampled from Tunisian coastal areas.

Seven (7) sites from to the Tunisian coastal area were monitored through four seasons using several oxidative stress biomarkers including lipid peroxidation. Catalase (CAT) specific activity was determined in clam Ruditapes decussatus digestive gland. Lipid peroxidation was assessed in the same tissues using malondialdehyde (MDA) concentration, neutral lipid (NL) and lipofuscin accumulation. Results show that catalase specific activity varies between sites and fluctuates in time. The highest CAT activities were recorded in sites from Bizerta Lagoon and sites neighbouring Sfax city. Malondialdehyde accumulation in digestive gland cells seems to be very pronounced in animals sampled in summer and autumn. Digestive gland sections of clams sampled from reported heavy metal polluted sites exhibited higher volume density of residual bodies. Our results showed that the neutral lipids and lipofuscin content in clam’s digestive gland are more sensitive to general stress as compared to other biomarkers, and could be used successfully in clams as early warning tools in field biomonitoring programs.

Comparative study of the bioaccumulation and elimination of trace metals (Cd, Pb, Zn, Mn and Fe) in the digestive gland, gills and muscle of bivalve Pinna nobilis during a field transplant experiment.

The purpose of this study was to investigate the long-term bioaccumulation and elimination of Cd, Pb, Mn, Zn and Fe by Pinna nobilis tissues after their 90 day-transplantation period at Téboulba fishing harbor. During the transplantation period, the Cd, Pb, Mn, Zn and Fe concentrations in the different tissues of the mussels were measured before and after exposure period. Metal (Cd, Pb, Mn, Zn and Fe) accumulation in P. nobilis mussels varied depending on the analyzed tissue and the caging times. Notable differences in Cd, Pb, Mn, Zn and Fe accumulation patterns within the digestive gland, gills and muscle were found and may be due to the ability of each tissue to accumulate metals. During the depuration phase, the elimination of Cd, Pb, Mn, Zn and Fe depended on the target tissue and the metal speciation. Cd, Pb, Mn and Fe were eliminated rapidly from one organ and increased in other when compared to those of 90 day transplanted mussels. The increase of metal loads during the elimination phase is not clear and particularly what kind of processes is responsible for such response. However, it is reasonable to assume that metals increase is related to the existence of an accumulation/detoxification mechanism, which involves the transport of metals from an organ to another. The data obtained indicate that because of the significantly high quantities of Cd, Pb, Mn, Zn and Fe accumulated during the exposure phase, the transplanted mussels are suitable bioindicators for monitoring trace metals in marine ecosystem.

Biochemical responses and metals levels in Ruditapes decussatus after exposure to treated municipal effluents.

This study assessed the responses of biochemical biomarkers and metals levels in Ruditapes decussatus exposed to the increasing concentrations of treated municipal effluents (TME) discharged into the Tunisian coastal area. Clams were exposed to 0%, 1%, 3% and 10% for 7 and 14 day and the following biochemical responses were measured: (1) catalase activity and lipid peroxidation levels (TBARS) as oxidative stress biomarkers, (2) gluthathione S-transferase (GST) activity as a phase II conjugation enzyme; (3) cholinesterase activity (ChE) as biomarker of neurotoxicity, and (4) metallothioneins as a proteins highly induced by heavy metals. A significant uptake of Cu, Cd and Zn in digestive gland and serious biochemical alterations were observed. Thus, exposure of clams to croissant concentration of TME have the potential to increase the oxidative stress biomarkers (TBARS, CAT activity) and MT levels; and decrease ChE activity in both gills and digestive gland. Current experimental results suggest that CAT, GST, ChE activities and MT and TBARs levels in gills and digestive gland of clam R. decussatus are sensitive and suitable responses for assessing the effects of anthropogenic contaminants on the aquatic ecosystems, particularly effluent complex mixtures.