Studies On The Methylation Of Inorganic Arsenic And Its Molecular Mechanism In The Clam Ruditapes Philippinarum

Arsenic(As) is a ubiquitous toxic metalloid, and mainly exists in the form of highly toxic inorganic arsenic in water. In view of the severe offshore arsenic pollution in our country, the bioaccumulation, biotransformation and detoxification of the clam Ruditapes philippinarum, which are widely distributed in near shore and estuarine areas and extensively used in biomonitoring programs, were investigated after waterborne exposure to arsenite(AsⅢ) or arsenate(AsⅤ). Techniques of analytical chemistry, molecular biology, and biochemistry, etc. were applied to study the bioaccumulation and biotransformation in tissues of R. philippinarum, and investigated the important role of Glutathione-S-transferase Ω(GST Ω) in the process of arsenic metabolism in the clams, and reveals the physiology and histopathological responses to arsenic exposure in the clams. The results were expected to not only provid an important basis for elcucidating the arsenic bioaccumulation and biotransformation mechanism in R. philippinarum, but also provide a theoretical basis for marine shellfish breeding and food safety. The main results were as follows:(1) Inorganic arsenic showed low bioavailability, and the bioavailability of As(Ⅲ) was slightly higher than As(Ⅴ) to the clams. Arsenic biotransformation in the clams might include As(Ⅲ) oxidation besides As(Ⅴ) reduction and subsequent methylation. The bioaccumulation and biotransformation for inorganic arsenic shows differences in digestive glands and gills of the clams, and arsenic was apt to be accumulated in digestive glands. Besides, the results demonstrated that arsenobetaine(AsB) and dimethylarsinic acid(DMA) was the major arsenic speciation in all treatments, and inorganic arsenic in gills was mainly converted to DMA, but AsB in digestive glands, indicating the different transformation efficiency in different tissues. Metallothionein-like proteins(MTLP) is the main binding site of arsenic at subcellular level, followed by cell debris in all treatments. Total As concentrations in metal sensitive fraction(MSF) changed more obviously, while total arsenic concentrations in biologically detoxified metal fraction(BDM) showed almost no change. And compared with the digestive glands, the gills tissues of the clams demonstrated limitied detoxification capability.(2) The recombinant GST Ω protein, the rate-limiting enzyme for biotransformation of inorganic arsenic, functioned as arsenate reductase, and expression of GST Ω enhanced the arsenic tolerance in the arsenate-sensitive strains E. coli AW3110(DE3)(ΔarsRBC), indicating its important role in biotransformation and detoxification of inorganic arsenic.(3) The expression of GST Ω was tested from the transcription level and protein levels respectively after inorganic arsenic exposure. After the inorganic arsenic exposure, the relative mRNA expression and arsenate reductase activity of GST Ω increased in digestive glands and gills of the clams in many treatments, further suggesting that GST Ω may play an important role in the process of inorganic arsenic methyl transformation and detoxification. Meanwhile, GST Ω was investigated by immunohistochemistry in the gills and digestive glands. The results showed that the GST Ω was mainly located in the epithelial cells of the gill filaments and the blood cells in the gill cavity, and mainly located in the epithelial cells of the digestive tubules.(4) After different concentrations of inorganic arsenic exposure for a period of time, GSH content, GR and GST activity of the clams in many treatments increased significantly, suggesting a resistance to oxidative stress. However, as exposure time increased, the gills, digestive glands and mantles had been histopathologically damaged to different extent. For example, after 100 μg/L As(Ⅲ) exposure for 30 days, the tubules of digestive glands were disfigured and necrotic, and fracture and distortion of muscle fiber was observed; epithelial cells lining at the gill filaments became disintegrated and vacuolated; inner and outer epithelial cells of mantles damaged severely. As(Ⅲ) exposure caused severer damage than As(Ⅴ) exposure, confirming that As(Ⅲ) is more toxic than As(Ⅴ).