Hamadi Boussetta

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.

Gene Expression Rhythms in the Mussel Mytilus galloprovincialis (Lam.) across an Annual Cycle

Seasonal environmental changes may affect the physiology of Mytilus galloprovincialis (Lam.), an intertidal filter-feeder bivalve occurring commonly in Mediterranean and Atlantic coastal areas. We investigated seasonal variations in relative transcript abundance of the digestive gland and the mantle (gonads) of males and females. To identify gene expression trends – in terms of relative mRNA abundance- we used a medium-density cDNA microarray (1.7 K probes) in dual-color competitive hybridization analyses. Hierarchical clustering of digestive gland microarray data showed two main branches, distinguishing profiles associated with the “hot” months (May–August) from the other months. Genes involved in chitin metabolism, associated with mussel nutrition and digestion showed higher mRNA levels during summer. Moreover, we found different gene transcriptomic patterns in the digestive glands of males when compared to females, during the four stages of mussel gonadal development. Microarray data from gonadal transcripts also displayed clear patterns during the different developmental phases respect to the resting period (stage I) with peak relative mRNA abundance at the ripe phase (stage III) for both sexes. These data showed a clear temporal pattern in transcriptomic profiles of mussels sampled over an annual cycle. Physiological response to thermal variation, food availability, and reproductive status across months may contribute to variation in relative mRNA abundance.

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.

Polychlorinated biphenyls (PCBs) and Polybrominated Diphenyl Ethers (PBDEs) in surface sediments from Monastir Bay (Tunisia, Central Mediterranean): occurrence, distribution and seasonal variations.

An assessment of PCB and PBDE contamination of surface sediments in Monastir Bay was carried out in two contrasted seasons of the year. Samples were collected from 5 sites and analyzed for the ∑7 marker PCBs (i.e. PCBs 28, 52, 101, 118, 138, 153 and 180) and ∑4 PBDE congeners (PBDEs 47, 99, 119 and 153) by GC/ECD. Concentrations of both PCBs and PBDEs showed seasonal variations. PCB concentrations were in the range of 3.1-9.3 ng g(-1) and 1.1-8.1 ng g(-1) in wet and dry season respectively, and sediments were considered moderately contaminated with PCBs. All PCBs analyzed were detected in surface sediments. PCB 153 and 52 congeners showed the highest relative abundance in both winter and summer. PBDE concentrations ranged from not detect to 0.1 ng g(-1), with only BDE-47 congener detected in sediments and only in winter. Analysis of spatial and seasonal variations indicated that PCB distribution is governed by hydrodynamics and temporal variability of inputs. While the PCB contamination appeared to be mainly land-based, PBDEs are suspected to originate from atmospheric deposition.

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.

Linking changes at sub-individual and population levels in Donax trunculus: Assessment of marine stress

Research in ecotoxicology currently focuses to fill the gap existing between sub-organismal responses (e.g. biomarkers) to toxicants and effects occurring at higher levels of biological organisation (e.g. population). The intra-sedimentary bivalve Donax trunculus commonly found in West Atlantic and Mediterranean coasts is a sentinel species useful for biomonitoring studies in sandy beaches. The objective of this work was to link responses at the infra-individual level (core biomarkers as early and sensitive tools) to supra-individual level (population for its ecological relevance). D. trunculus, originating from a polluted site (Radès Méliane) and a comparatively reference site (Sidi Jehmi) in the Gulf of Tunis (Tunisia), were collected bimonthly from November 2008 to October 2009. An increase in catalase activities was usually observed in bivalves from the polluted site compared to the reference site whereas no differences in TBARs were depicted. The anti-oxydant enzyme (catalase) could be able to prevent the deleterious effect on the lipid membranes. Usually GST activities were decreased in the polluted site. Significantly high inhibition in AChE activities in bivalves from the polluted site suggested neurotoxicity disturbances to their in situ exposure to compounds such as organophosphate and carbamates pesticides, heavy metals. Size-distribution of populations of D. trunculus from the polluted Radès Méliane site consisted of four cohorts whereas five cohorts were depicted in the comparatively reference Sidi Jehmi site. The mean total length size and the growth rate of cohorts were significantly reduced in the impacted site compared to the reference site. In conclusion, it may be suggested that disturbances in responses to chemical stress at the infra-individual level could be linked to the responses observed at the population level.

Transcriptional expression levels and biochemical markers of oxidative stress in Mytilus galloprovincialis exposed to nickel and heat stress.

The present study aims to evaluate transcriptional expression levels and biochemical markers of oxidative stress responses to nickel (Ni) exposure along with heat stress gradient in a mussel (Mytilus galloprovincialis). For this purpose, we investigated the response of oxidative stress markers, metallothionein accumulation and gene expression in digestive gland of mussels exposed to a sublethal concentration of Ni (2.5μM) along with a temperature gradient (18°C, 22°C, and 26°C) for 24h and 72h. Ni digestive gland uptake was evaluated after the exposure periods. Co-exposure to Ni and higher temperature (26°C) for 72h significantly decreased the antioxidant enzyme activities termed as catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) and caused a pronounced increase of lipofuscin and neutral lipid (NL) accumulation. Ni-uptake was different with respect to the exposure periods and temperatures in Ni-exposed mussels. Sod, cat, gst, mt-10 and mt20 gene expression levels showed a substantial increased pattern in animals exposed for one day to heat stress compared to the control condition (18°C). The same pattern but with highest level was registered in animals co-exposed to Ni and temperatures within one day. Three days exposure to 18°C, 22°C and 26°C, resulted in a significant decrease in mRNA abundance of cat, gst and sod and a significant down-regulation of mts targets (22°C and 26°C). Our data provide new insights into the importance of the early protective response of oxidative stress related-gene expression and regulation in mussels challenging heat stress and sublethal Ni concentration.

Evaluation of enzymatic biomarkers and lipoperoxidation level in Hediste diversicolor exposed to copper and benzo[a]pyrene.

This study aims to evaluate the effects of exposure to copper, benzo[a]pyrene, and to their mixture on enzymatic and lipid peroxidation biomarkers in Hediste diversicolor. Worms were submitted to 1 microM of both single compounds and to their mixture during a period of test of 12, 24, 36, and 48 h. The biomarkers selected in this work were the activities of cytochrome P450-dependent NADPH cytochrome c reductase (NADPH red) as phase I enzyme, glutathione-S-transferase (GST) as phase II enzyme, and the acetylcholinesterase (AChE) activity as neurotoxicity marker. Oxidative stress was evaluated using catalase activity (CAT) and malondialdehyde accumulation (MDA). The NADPH red activity was not significantly affected by copper exposure; it shows a drastic increase in both B[a]P and mixture-exposed organisms. GST activities were significant in B[a]P-exposed worms only after 36 h, and in animals exposed to the mixture after 12 and 48 h. The ACHE activity was inhibited only in B[a]P-exposed worms.