| The occurrence of toxic cyanobacterial blooms in eutrophic lakes, reservoirs, and recreational waters has become a worldwide problem, and there is recent concern that cyanotoxins that may enter drinking water and pose a threat to public health. Several cyanobacterial species, specifically Microcystis aeruginosa, may produce a variety of potent toxins, including hepatotoxins. Among the hepatotoxins, cyclic peptides known as microcystins (MCs) are the predominant toxins in freshwaters worldwide. Among the MCs, the most common, and also the most extensively studied, is microcystin-LR (MC-LR). MC-LR can inhibit the phosphatases 1 and 2A(PP1, PP2A). It has been well documented that MC-LR may have adverse effect on embryo development and growth of fish, and pathological damages in liver and kidney of fish, and oxidative damage in fish exposed to MC-LR.The liver is main target organ of MC-LR, and it plays an important role in detoxification against toxin. Although, it was believed that metabolism of MC-LR begins with a conjugation reaction to glutathione (GSH) catalyzed by glutathione S-transferases (GST), resulting in toxicity of MC-LR lower, and organism could promote the antioxidative stress response against oxidative damage owing to MC-LR; several previous studies investigated the mechanism of degradation and resistance of MC-LR in terms of pathology and physiology of organisms, while there is little information on the changes of gene expression and the regulatory mechanism of related detoxification genes in fish feeding on phytoplankton when they are exposed to MC-LR, including the differential expression of detoxification-related genes of bighead carp treated with MC-LR. Bighead carp of exposure group were injected intraperitoneally with commercial MC-LR(purity>95%) at dose (200 g/kg, BW), and the control group of four fish was injected with 0.8% sterile saline. Then, Four fish in both groups were killed at 0.5, 1, 3 and 5 h post-injection. The initial time of GST gene expression was selected as a critical detoxification-related genes indicator and semi-quantitative RT-PCR was performed to determinate which hour sample was used to constructe the SSH library. The results suggested that the transcriptional expression of the GST gene was significantly decreased at 0.5 h post-injection, and the response of detoxification to MC-LR had been occurring. So mRNA of liver sample from 0.5 h exposed to MC-LR and the control were applied for reverse transcription and construction of a cDNA library in SSH. In the present study , the forward subtracted library(control group as tester, exposure group as driver ) and the reverse subtracted library(exposure group as tester, control group as driver) were constructed, screening differentially expressed genes involved in apoptosis, signal transduction, cytoskeletal remodel, immune-related, material and energy metabolism which were extensively discussed, and these functional genes play important roles in declining the toxity of MC-LR and protecting organism from toxic damage. However, the immune-related genes from the forward subtracted library are different with the reverse subtracted library, which are responsible for the diverse functions of immunity system.The lactate dehydrogenase and head shock protein 70 gene from forward subtracted library upregulated play a crucial role in antioxidative response against MC-LR in bighead carp, especially the HSP70, the induction and upregulation of HSP70 has been shown to provide cells with stress tolerance to subsequent damage owing to MC-LR. Additionally, the presence of serine (or cysteine) proteinase inhibitor in forward subtracted library suggested the mechanism of toxication of MC-LR is that phosphatases 1 and 2A were inhibited. The apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library, which are closely related to the cell apoptosis and tumor formation. Fs309 and Fs314 EST from the forward subtracted library, with homology to S-adenosylhomocysteine hydrolase and cytochrome P450 gene, respectively, were observed to increase significantly compared to the control group. The proteins coded by the two genes represent one of the body's most important mechanisms of defense against chemical-induced toxicity. Additionally, the expression of Rs204 and Rs252 were down regulated, and the results provide a strong argument for the high validity of the SSH libraries. Additionally, the S-adenosylhomocysteine hydrolase and cytochrome P450 genes are more sensitive in the toxicity response to MC-LR than GST, so these are the two candidate genes to be considered as the indicators of this response to toxicity of MC-LR. |
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