METALLOTHIONEIN GENE EXPRESSION AND PROTEIN LEVELS IN TRIPLOID AND DIPLOID OYSTERS CRASSOSTREA GIGAS AFTER EXPOSURE TO CADMIUM AND ZINC (original) (raw)
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Marine Environmental Research, 2019
The metal-binding protein metallothionein (MT) is widely used as a biomarker of metal contamination. In this study, we cloned a MT gene (sgMT) from the Sydney rock oyster Saccostrea glomerata. The gene encodes a MT-I protein with a classical αβ domain structure and is expressed as two transcripts resulting from alternative polyadenylation. The gene promoter contains two putative metal-responsive elements (MREs) which are known to be required for metal-inducible transcription. A specific and efficient qPCR assay was developed to quantify sgMT mRNA expression. Further, we assessed whether prior metal exposure history influences sgMT mRNA expression upon subsequent metal exposure. Oysters with varying prior metal exposure histories (contaminated and reference) were exposed to Cu, Cd and Zn. Expression of sgMT generally increased with metal dose, and oysters with an elevated past metal exposure history exhibited higher sgMT expression under Cd and Zn stress, representing a potential acclimatory response to prior metal exposure.
Dose-Dependent Effects of Metals on Gene Expression in the Sydney Rock Oyster, Saccostrea glomerata
In the current study, we tested the effects of common environmental contaminants (the metals zinc and lead) on gene expression in Sydney rock oysters (Saccrostrea glomerata). Oysters were exposed to a range of metal concentrations under controlled laboratory conditions. The expression of 14 putative stress response genes was then measured using quantitative, real-time (q) PCR. The expression of all 14 genes was significantly affected (p < 0.05 vs. nonexposed controls) by at least one of the metals, and by at least one dose of metal. For 5 of the 14 target genes (actin, calmodulin, superoxide dismutase, topoisomerase I, and tubulin) the alteration of expression relative to controls was highest at intermediate (rather than high) doses of metals. Such responses may reflect adaptive (acclimation) reactions in gene expression at low to intermediate doses of contaminants, followed by a decline in expression resulting from exposure at higher doses. The data are discussed in terms of the intracellular pathways affected by metal contamination, and the relevance of such gene expression data to environmental biomonitoring.
Journal of Experimental Biology, 2008
Cadmium (Cd) is an important toxicant in estuarine and coastal environments that can strongly affect energy balance of aquatic organisms by increasing the organismʼs basal energy demand and reducing its aerobic capacity. Mechanisms of cadmiuminduced increase in basal metabolic costs are not well understood and may involve elevated detoxification costs due to the synthesis of cellular protective proteins and glutathione. We studied the short-term effects of cadmium exposure (4·h) on protein and glutathione (GSH) synthesis and expression of stress proteins (heat shock proteins HSP60, HSP70 and HSP90) and metallothioneins in isolated gill and hepatopancreas cells of the eastern oyster, Crassostrea virginica. Our study showed that exposure to cadmium resulted in a dose-dependent increase in the rate of protein synthesis in oyster cells, which reached 150% of the control at the highest tested Cd level (2000·mol·l -1 ). GSH synthesis was significantly inhibited by the highest Cd concentrations, especially in hepatopancreas, which resulted in a slight but significant decrease in the total GSH concentrations. Elevated protein synthesis was associated with the increased expression of metallothioneins and heat shock proteins. Interestingly, stress protein response differed considerably between gill and hepatopancreas cells. In hepatopancreas, expression of metallothionein mRNA (measured by real-time PCR) increased 2-8-fold in response to Cd exposure, whereas no significant increase in metallothionein expression was found in Cd-exposed gill cells. By contrast, HSP60 and HSP70 protein levels increased significantly in Cd-exposed gill cells (by 1.5-2-fold) but not in hepatopancreas. No change in HSP90 expression was detected in response to Cd exposure in oyster cells. These data indicate that metallothionein expression may provide sufficient protection against Cd-induced damage to intracellular proteins in hepatopancreas, alleviating the need for overexpression of molecular chaperones. By contrast, Cd detoxification mechanisms such as inducible metallothioneins and GSH appear to be insufficient to fully prevent protein damage in gill cells, thus necessitating induction of HSPs as a secondary line of cellular defense. Therefore, gills are likely to be among the most Cd-sensitive tissues in oysters, which may have important implications for impaired oxygen uptake contributing to energy misbalance and reduced aerobic scope in Cd-exposed oysters.
Marine Pollution Bulletin, 2017
This study was designed to identify in the pearl oyster Pinctada margaritifera, used as a bio-accumulator, molecular biomarkers for the presence of heavy metals in the lagoon environment. Pearl oysters were exposed to 2 concentrations (1 and 10 μg L− 1) of cadmium (Cd) and chromium (Cr) compared to a control. Twelve target genes encoding proteins potentially involved in the response to heavy metal contamination with antioxidant, detoxification or apoptosis activities were selected. P. margaritifera accumulated Cd but not Cr, and mortality was related to the amount of Cd accumulated in tissues. In response to Cd-Cr contamination, metallothionein (MT) was significantly up-regulated by Cd-Cr at both concentrations, while 7 others (SOD, CAT, GPX, GSTO, GSTM, CASP, MDR) were down-regulated. Based on the development of these molecular tools, we propose that the pearl oyster, P. margaritifera, could be used as a sentinel species for heavy metal contamination in the lagoons of tropical ecosystems. Highlights ► The pearl oyster Pinctada margaritifera accumulated cadmium. ► Metallothionein gene expression was significantly up-regulated by cadmium. ► P. margaritifera could be a sentinel species of the heavy metals contamination.
Fundamental and Applied Toxicology, 1996
An acute (48-hr) lethal toxicity study of turbot at various stages from hatch to juveniles showed that prior to first exogenous feeding, larvae of turbot were an order of magnitude more sensitive to cadmium toxicity than later developmental stages (L.C50 of 0.18-0.23 ppm Cd versus 2-5 ppm Cd). To investigate the possible role of metallothionein (MT) in Cd tolerance, the concentrations of MT mRNA were determined in control and Cd-exposed animals using a plaice MT cDNA probe. MT mRNA was expressed throughout embryonic and early larval development at levels approximately twice those in juvenile turbot liver apart from the period around hatching, when there was a decreased level of expression. During endogenous feeding, despite larval drinking, MT mRNA levels did not appear to be inducible by exposure to Cd in the water within 3 days of hatching but were induced three-to fivefold after 48 hr exposure to waterborne Cd at all later stages of development. The results suggest that Cd tolerance is associated with metal induction of MT gene transcription and that the enhanced sensitivity of very early stage larvae is attributable to a deficiency in MT gene transcription, c 1996 soday of Toxi col ogy
Marine Ecology Progress Series, 2003
Metallothioneins (MTs) are small essential proteins involved in cellular processes of metal handling and detoxification. Here, we describe the structure of 2 MT genes in a marine mollusc, the European flat oyster Ostrea edulis. The sequences of these 2 genes code for 2 proteins of 74 and 71 amino acids, respectively, with the 71 amino acid protein having an abnormal lack of cysteine residues. The direct quantification of metal ions bound to purified recombinant proteins demonstrated that the metal-binding capacity of the 2 proteins differs by about 20%. An enzyme-linked immunosorbent assay (ELISA) test, used to quantify MT protein expression in O. edulis exposed to metal under laboratory conditions, showed no significant induction of MTs, either in the gills or the digestive gland despite an increase in metal concentration observed in the same tissues. Similar results were observed with an MT-RNA expression study. These results seem to indicate a low involvement of MTs in metal detoxification in O. edulis. Examination of polymorphism in the coding sequence of OeMT (O. edulis metallothionein) genes by single-strand conformation polymorphism (SSCP) revealed a lack of genetic diversity in field populations.
BioMetals, 2006
Metallothionein (MT) response to cadmium (Cd) and zinc (Zn) bioaccumulation after single or combined direct exposure was compared in two freshwater bivalves, Dreissena polymorpha (zebra mussel) and Corbicula fluminea (Asiatic clam). Bivalves were exposed to 0.133 lM Cd and/or 15.3 lM Zn, with metal and MT concentrations analysed in the whole soft body after 1, 3, 10 and 24 days of exposure and compared with controls. Results showed significant increase in MT concentrations in both species exposed to Cd and Cd+Zn with a higher accumulation of the protein compared to the control in D. polymorpha for nevertheless similar Cd levels accumulated with time. Exposure to Zn alone led to a significant increase in MT concentrations only in C. fluminea, whereas there was a lack of MT gene induction in the zebra mussels which was confirmed by MT mRNA quantification in gills (RT-PCR). Mussel mortality after 10 days of exposure to Zn and Cd+Zn is discussed with regard to detoxification mechanisms, which include metallothioneins.
Aquatic Toxicology, 2006
Turbot (Scophthalmus maximus) were exposed to two sublethal concentrations (1 and 10 mg metal/l) of cadmium (8.9 and 89 M Cd), copper (15.26 and 152.6 M Cu) and zinc (15.3 and 153 M Zn) for 7 days, and afterwards were maintained depurating for 14 days. Immunoreactive metallothioneins (irMTs) and metal ions were localized in the branchial epithelium by immunohistochemistry (using an anti-Cod MT antibody) and autometallography (AMG), respectively. Metal ions were demonstrated by AMG as black silver deposits (BSD), mainly in mucocytes (MC) and to a lesser extent in the other branchial cell-types (respiratory cells (RC), chloride cells (CC) and basal layer cells (BLC)). Irrespective of the metal supplied, BSD were rapidly visualized in MC after 1 h of exposure. This accumulation did not increase with increasing exposure time and concentration. Metallothionein expression was mainly observed in mature CC in the interlamellar space for all exposure conditions and it was shown that all mature cells express the same amount of irMT. The number of CC exhibiting irMT in metal-exposed turbots increased following short exposure times (1 h-1 day) in the filament epithelium and following longer exposure times (1-7 days) in the secondary lamellae. Total levels of irMT in the gills (quantified by image analysis and densitometry) increased significantly in metal-exposed turbot and were related to increased exposure times. It can be concluded that the total content of irMT in the gills of metal-exposed turbot is governed by changes in the number of mature CC expressing the protein. The quantification of total irMT in branchial CC can be considered as a reliable biomarker of metal exposure since reflects changes in metal bioavailability. This approach based on cell-selective immunohistochemistry can be simplified by only quantifying the number of mature CC. In addition, the dramatic increase of CC in the gills that produces epithelial thickening of the FE enhances migration of CC up to the edge of the SL and provokes the hypertrophy and fusion of secondary lamellae can be considered as unspecific biomarkers of effect indicating disturbed health in turbot.
Aquatic toxicology, 2003
A semi-quantitative RT-PCR protocol was developed to directly evaluate metallothionein (MT) mRNA expression in different tissues of mangrove oysters (Crassostrea rhizophorae ), using b-Actin (ACT) as a normalizing gene. Clones with high degree of identity from partial coding sequences were obtained for both MT and ACT. Although not statistically significant, high relative accumulation of MT mRNA was observed in the digestive gland (DGG), but not in the gills, from samples collected from both control and contaminated sites. Nevertheless, MT expression was not comparable to the high levels of metal in the contaminated oysters. Results indicate that the variation in relative MT mRNA levels from different samples of the same site could be due to multiple gene copies or different MT isoform induction. #