Metallothionein as a biomarker for mercury contamination in mussel, Perna viridis

Abstract

Mercury (Hg) is one of the most toxic heavy metals. Its discharges to the marine environment are of great concern. Very low level of Hg still have chronic effect to many living organisms and sophisticated equipment are needed for the measurement at such low concentration. Metallothioneins (MT) are low molecular weight cytosolic proteins found in eukaryotic species. They are capable of binding a variety of heavy metals, including Hg. The study focused on the effects of the various levels of dissolved mercury to the level of metallothionein in mussels, Perna viridis. The experiment was designed to verify the methods, effectiveness for the quantification of metallothioneins in mussel. The results show that semi-quantitative RT-PCR is more promisingtechnique than gel electrophoresis and silver staining in measuring of mussel MT mRNA and eventually this technique can be applied for the assessment of mercury contamination in mussels. The study focused on the digestive tract of the mussels which has been shown to play an important role in tissue distribution of MT. Total RNA from the digestive tract of the mussels exposed to various levels of mercuric chloride was extracted and converted to cDNA by reverse transcription. Oligonucleotide primers were designed to amplify a 220 bp segment of the mussel MT cDNA. The expression of Beta-actin was used as reference. The RT-PCR data showed that there was a significant different between treatments and control in digestive tract MT mRNA in subjects within 2 to 4 weeks of mercury exposure (p < 0.05). At concentration of 1 mg/L HgCl2 induced the MT mRNA levels significantly comparing with control. Moreover, this study also investigated the variation of metallothionein forms in mercury-exposed mussels. Six isoforms were identified by single strand conformation polymorphism (SSCP) method. This data suggests that MT mRNA levels of digestive gland from mussel respond to mercury exposure and the response could serve as a more useful assessment of mercury contamination in marine environment.