Proteomic approach for identifying gonad differential proteins in the oyster (Crassostrea angulata) following food-chain contamination with HgCl2.

UNLABELLED: Hg discharged into the environmental waters can generally be bioaccumulated, transformed and transmited by living organisms, thus resulting in the formation of Hg-toxicity food chains. The pathway and toxicology of food chain contaminated with environmental Hg are rarely revealed by proteomics. Here, we showed that differential proteomics had the potential to understand reproduction toxicity mechanism in marine molluscs through the Hg-contaminated food chain. Hg bioaccumulation was found in every link of the HgCl2-Chlorella vulgaris-oyster-mice food chain. Morphological observations identified the lesions in both the oyster gonad and the mice ovary. Differential proteomics was used to study the mechanisms of Hg toxicity in the oyster gonad and to find some biomarkers of Hg contamination in food chain. Using 2-DE and MALDI-TOF/TOF MS, we identified 13 differential protein spots, of which six were up-regulated, six were down-regulated, while one was undecided. A portion of these differential proteins was further confirmed using real-time PCR and western blotting methods. Their major functions involved binding, protein translocation, catalysis, regulation of energy metabolism, reproductive functioning and structural molecular activity. Among these proteins, 14-3-3 protein, GTP binding protein, arginine kinase (AK) and 71kDa heat shock connate protein (HSCP 71) are considered to be suitable biomarkers of environmental Hg contamination. Furthermore, we established the gene correspondence, responding to Hg reproductive toxicity, between mouse and oyster, and then used real-time PCR to analyze mRNA differential expression of the corresponding genes in mice. The results indicated that the mechanism of Hg reproductive toxicity in mouse was similar to that in oyster. We suggest that the proteomics would be further developed in application research of food safety including toxicological mechanism.

BIOLOGICAL SIGNIFICANCE: It is well known that mercury (Hg) is one of the best toxic metal elements in nature. The research reports as previously described indicated that multiple mercury compounds can directly contaminate the aquatic animals by flowing of water body and through the diffusion of air. The pollution sources of the mercury compounds in marine water were mainly found from the pathways such as steam power plant and mineral exploitation which are located on the inshore. Of note, after being released into environmental waters, mercury compounds undergo the processes of bioaccumulation, transformation and transmission in living organisms, thus resulting in the multiple forms of Hg found in Hg-toxicity food chains, and among them, methyl mercury (MeHg) showing the high toxic characteristics is the main form of Hg. The abundant reports indicated that the metal salts were easily found within the various organs of the animals, but it is difficult to judge the level of its perniciousness according to its content only in vivo. Here, the algae to have been contaminated by the mercury compounds have the ability for contaminating both the fish and shellfish as food pathway quickly. If these fish and shellfish edible as food will be taken by human, they will further affect the human health badly. However, studies about their perniciousness are rarely reported, especially in using proteomics. The oysters as normal food are largely consumed in Southern China, especially in Xiamen City. Similarly, a pathway question that the contaminated oysters can effect on the human health such as cancer is unclear or poorly understood. Here, we showed that an analytical technology such as differential proteomics has potential to understand toxicity mechanism induced by Hg-contamination through the food pathway. It is for reason that the oyster proteomics including relative analytical methods have been used to reveal the contaminant level and to determine its perniciousness using toxic algae as food. Here, we also indicated that the research here shows great significance for both analysis of food safety and toxicology of the metal compounds. In addition, a few biomarkers have shown their strong potential for monitoring the level of Hg pollution in sea in the manuscript and gene correspondence between mouse and oyster, the two contiguous links of the Hg-contaminated food chain, was further investigated to better illustrate our finding in the analysis of food chain proteomics. Moreover, similar research work is rarely reported compared to the current proteomic development, showing that a lot of novel results by proteomic methods in the manuscript have strong potential for developing the new area of food chain proteomics.