| In view of heavy metal pollution in coastal waters and the deterioration of shellfish resources in China, the response mechanism of Mactra veneriformis, the infaunal suspension-feeding bivalve, to cadmium (Cd) and mercury (Hg) was studied. The full-length cDNAs of metallothionein (MT), superoxide dismutase (SOD), and lysozyme (LYZ), which are defense components, in M. veneriformis were cloned. Furthermore, mRNA expression and protein content (enzyme activity) of MT, SOD, and LYZ were determined under Cd, Hg, and Vibrio exposure. Besides, the genotoxicity of Cd and Hg was assessed.1. The full-length cDNAs of MvMT, MvSOD, and MvLYZ contained 830, 689, and 808 nucleotides encoding 59, 159, and 194 amino acids, respectively. The deduced amino acid sequence of MvMT included conserved structural patterns of invertebrate MT and mollusk MT with high cysteine content (30.5%) and without aromatic and histidine residues. Two Cu-Zn SOD family signature sequences were found in MvSOD deduced amino acid sequence, in which the residues required for copper (His-51, -53, -68, and -125) and zinc (His-68, -76, and -85 and Asp-88) binding were well conserved. Two conserved active sites (Glu89and Ser105) and a highly conserved sequence of I-type LYZ near the active sites were detected in MvLYZ deduced amino acid sequence. Multiple alignments revealed that deduced amino acid sequences of MvMT, MvSOD, and MvLYZ had high similarity with the corresponding sequences of other mollusks.2. Clams were exposed to Cd (50, 100, and 200μg/L) or Hg (10, 20, and 40μg/L) for 21 days. MvMT and MvSOD mRNA transcripts in the digestive gland, gill, and mantle of M. veneriformis could response to Cd or Hg exposure in a tissue-specific way. In the digestive gland, Cd treatment significantly elevated MvMT and MvSOD mRNA expression in a dose-dependent manner and elevated MT protein level in a dose- and time-dependent manner. SOD activity significantly increased at the start of Cd exposure, then decreased and finally returned to the normal level. A dose-dependent increase of MvMT and MvSOD mRNA expression in 7 days at the beginning of exposure, stimulation of MT protein, and alteration of SOD activity were observed under Hg stress. These results suggested that MT and SOD cooperated in resisting Cd and Hg toxicity and maintaining cellular metabolic homeostasis in M. veneriformis. MT and SOD mRNA expressions have great potential as biomarkers of Cd and Hg pollution in the aquatic environment for the studied species.3. MvMT, MvSOD, and MvLYZ mRNA expression in the digestive gland of M. veneriformis showed significant alteration under heavy metals (200μg/L Cd + 20μg/L Hg) and/or MM21 Vibrio parahaemolyticus (1.2×1010CFU/L) exposure for 7 days. The effect on the mRNA expression caused by combined exposure was not the simple superposition of that caused by single exposure. The data indicated that the regulation of MvMT, MvSOD, and MvLYZ mRNA expressions under combined exposure was complex. In view of the high mortality of M. veneriformis under combined exposure, it was inferred that the combined exposure had a more severe impact on M. veneriformis, which reduced the physiological function of the animal.4. There was no obvious DNA damage in haemocytes caused by 50, 100, and 200μg/L Cd during the exposure period. However, Hg caused significant DNA damage in haemocytes at doses of 10, 20, and 40μg/L in a dose- and time- dependent manner. Combination of 200μg/L Cd and 20μg/L Hg exposure induced a time-dependent increase of DNA damage. The degree of DNA damage in the combined exposure group was severer than that in Hg-treated groups. These results indicated that the ranking of genotoxic potential to M. veneriformis was in decreasing order: Hg>Cd. There was a synergistic effect to DNA damage between Cd and Hg.
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